Method of producing riboflavyl phosphates



Patented Sept. 9, 1952 METHOD or PRODUCING mBoFLAvYt PHOSPHATES Leo A. Flexser, Upper Montclair, and Walter G.

.Farkas, Nutley, N. J., assignors to-Hofimanh- La Roche Inc., Nutley, N. J., a corporation of New J ersey i No Drawing. Application January 26,1951,

Serial No. 208,084

This invention relates to the manufacture of alkyl derivatives of riboflavin monophosphoric ticularly, the invention is concerned with lower alkyl riboflavyl phosphates, i. e., monoand dilower alkyl riboflavyl phosphates, which are new and useful compounds. We have also found that by subjecting the lower alkyl riboflavyl phosphates to hydrolysis, hydrolysis occurs preferentially at the lower alkyl groups rather than at the riboflavyl group; and that substantially pure riboflavyl phosphate is produced. The new lower alkyl riboflavyl phosphates are, accordingly, useful for the preparation of riboflavin monophosphoric acid ester, which is a soluble form of vitamin B2 and is of importance for its physiological activity. The mono lower alkyl riboflavyl phosphates hydrolyze with one molecule of water to give the corresponding lower alkanol and'riboflavyl phosphate. In the case of the dilower alkyl riboflavyl phosphates, two molecules of water are required for the hydrolysis. The hydrolysis is preferably carried out in the presence of a mineral acid, e. g., hydrochloric acid. Moderate warming facilitates the hydrolyzing action. By the hydrolysis, substantially pure riboflavin phosphate, free from lower alkyl phosphates and riboflavin, is readily prepared.

In general, the lower alkyl riboflavyl phosphates are prepared by reacting riboflavin with a lower alkyl chlorophosphoric acid ester, i. e., a mono lower alkyl dichlorophosphate or a dilower alkyl monochlorophosphate. When a mono lower alkyl dichlorophosphate is employed in the reaction with riboflavin, there appears to be first produced an intermediate ester hydrochloride salt, namely, a mono lower alkyl riboflavyl chlorophosphate hydrochloride, which, after mild hydrolysis, yields a corresponding mono. lower alkyl riboflavyl phosphate. When a dilower alkyl monochlorophosphate is employed, there is probably first produced an intermediate ester hydrochloride salt, namely, a dilower alkyl riboflavyl phosphate hydrochloride. The above is based on What we consider tobe the most probable course of the reaction with the monoand dilower alkyl chlorophosphates, it being understood, however, that we do not wish'to limit our invention to any speciiictheory of reaction.

As stated above, the monoand dilower alkyl riboflavyl phosphates can be further hydrolyzed to riboflavin phosphate. Such hydrolysis can 11 Claims. (o1.2so-zu.3)

preferably be carried out in the presence of a mineral acid. Since the complete hydrolysis to riboflavin phosphate can be carried out directly on the reaction mixture of "riboflavin and the lower alkyl chlorophosphates, it is not necessary to isolate the intermediate ester hydrochloride salts or the mono- '01 dilower 'alkyl riboflavyl phosphates from the reaction mixture. "In this case, the reaction mixture is sufficiently acid, so that further acid need not'be added to iacili= tate the hydrolysis, the addition of water alone being sufiicient to effect the hydrolysis.

In the preferred mode of practicing our process, we prepare the mono lower alkyl dichloro phosphate and the dilower alkyl monochlorm phosphate prior to reacting them with the ripeflavin. This is done by reacting phosphorus oxychloride with about one to two moles of a lower alkanol, e. g., methanol or ethanol. When one mole of the lower alkanol is reacted with phosphorus oxychloride, the mono lower alkyl dichlorophosphate is obtained. When two moles of the lower alkanol are reacted with phosphorus oxychloride, the dilower alkyl monochlorophosphate is obtained. These compounds need not be isolated from the reaction mixture, the riboflavin being merely added thereto for conversion to the corresponding lower alkyl riboflavyl phosphates.

Although it is preferred to prepare the lower alkyl chlorophosphates by prior reaction of phosphorus oxychloride with about one to two moles of the lower alkanol before addition of the riboflavin, it is to be understood that this order of addition of reactants is not critical. The preparation of the lower alkyl riboflavyl phosphates, may, for example, also be accomplished by admixture of the riboflavin and phosphorus oxychloridefollowed by careful addition of the lower alkanol. When the method is carried out in this way, practically .no reaction occurs between the riboflavin and the phosphorus oxychloridealone. However, when the lower alkanol is added, hydrogen chloride is evolved and the lower alkyl chlorophosphate is formed which then reacts upon the riboflavin. This procedure is attended by the possibility that some destruction of riboflavin may occur as considerable heat is generated when the alkanol contacts and reacts with the phosphorus oxychloride. Accordingly, it is preferred to react the alkanol and phosphorus oxychloride before the addition of the riboflavin in order to eliminate the destruction of the latter by local overheating. If, in addition, several hours are allowed to elapse water.

3 before riboflavin is added, a better yield of the lower alkyl riboflavyl phosphate is obtained, since the lower alkyl chlorophosphate will then be given sufficient time to be formed in optimal quantities for reaction.

The following examples will serve to illustrate the practice of our invention. It will be understood that the examples are illustrative only and are not intended to restrict the scope of the invention.

EXAMPLE I MOTZOWZBthl/l riboflaoyl phosphate To 36.6 cc. of phosphorus oxychloride (0.4 mole) were added slowly with stirring and cooling to room temperature 16.2 cc. of methanol (0.4 mole). After evolution of HCl gas had subsided, the reaction mixture, which contained 'mononiethyl dichlorophosphate, was allowed to stand for about 16 hours. 3.76 grams of riboflavin (0.01 mole) were added. Rapid solution took place andthe solution was stirred for hours at roomtemperature. It was then poured with cooling to room temperature into 500 cc. of anhydrous ether. The resulting yellow precipitate of the intermediate ester salt (monomethyl ri'boflavyl chlorophosphate hydrochloride) was filtered off, washed with ether and dried in vacuo. The-'dry'substance was dissolved in a mixture of 36- cc."v of dioxane and 4 cc. of

Shortly after completesolution was obtained, crystallization of monomethyl riboflavyl phosphate occurred. -The product was filtered off; washed with dioxane and ether, and dried. On potentiometric titration with sodium hydroxidea single inflection point, occurring at pH 5.0, was found.

EXAMPLE II Riboflavin monophosphoric acid ester The inoncrnethyl riboflavyl phosphate obtained in Example I was dissolved in 5 cc. of concen- .trated (36 per cent) hydrochloric to which were subsequently added 30 cc, of water. A syrup thereupon precipitated which crystallized on stirring. The riboflavin monophosphoric acid ester thus obtained was filtered off, washed with ethanol, and dried.

EXAMPLE III Dimethyl riboflavyl phosphate To 366 cc. of. phosphorus oxychloride were added with cooling to room temperature and stirring 32.4 cc.- of methanol (0.8 mole). After evolution-of HCl gas had subsided, the reaction product, which contained dimethyl monochlorophosphate, was allowed to stand for about 16 hours. 3.76 grams of riboflavin were then added. Rapid solution tool; place, and the solution was stirred at room temperature for 6 /2 hours. It was then poured with cooling into 500 cc. of anhydrous ether. The intermediate ester salt (dimethyl riboflavyl phosphate hydrochloride) separated as a syrup which was washed with ether. The syrup, upon treatment with ice, formed dimethyl riboflavyl phosphate which crystallized. The resulting slurr Was filtered, the crystals washediwith water, alcohol and ether, and dried in vacuo. The dimethyl riboflavyl phosphate thus obtained showed no acidic properties, being the ncutral phosphate ester. EXAMPLE IV Riboflavinmonophosphoric acid ester 0.5 gram. of dimethyl riboflavyl phosphate obtained as in Example III was dissolved in one cc. of concentrated hydrochloric acid and the solution diluted with live volumes of water. The product which crystallized was riboflavin monophosphoric acid ester.

EXAMPLE V Monoethyl riboflcwyl phosphate To 36.6 cc. of phosphorus oxychloride were added slowly with stirring and cooling to room temperature 23.3 cc. of anhydrous ethanol (0.4 mole). After evolution of HCl gas had subsided, the reaction mixture, which contained monoethyl dichlorophosphate, was allowed to stand for about 16 hours. 3.76 grams of riboflavin were then added. Rapid solution occurred, and the resulting solution. was stirred for 7 /2 hours at room temperature. It was then poured with cooling into 500 cc. of "dry ether. The resulting precipitate of intermediate ester salt (monoethyl riboflavyl chlorophosphate hydrochloride) was filtered, washed with ether, and driedin vacuo. The dried substance was dissolved in a mixture r of 36 cc. of dioxane and 4 cc. of water.- After 20 minutes, crystallization of monoethylriboflavyl phosphate occurred; The product was filtered off, washed with dioxane and ether, and

dried. Potentiometric titration with sodium hydroxide showed a single inflection point at about EXAMPLE VI Dicthyl riboflaoyl phosphate To 36.6 cc. of phosphorus oxychloride were added slowly and with stirring'qdfi cc. (0.8 mole) of anhydrous ethanol while maintaining the temperature at 20-25 C. After evolution of HCl gas had subsided, the reaction mixture, which contained diethyl monochlorophosphate, was permitted to stand for about 16 hours. 3.76 grams of riboflavin were then'added and the resulting mixture was stirred and warmed to 50 EXAMPLE VII 7 To 36.6 cc. of phosphorus oxychloride were added slowly and with stirring 46.6 cc. of anhydrous ethanol while maintaining the temperature at 20-25 C. After evolution of HCl gas had subsided, the reaction mixture, which contained diethyl monochlorophosphate, was permitted to stand for about 16 hours. 3.76 grams of riboflavin were then added and the resulting mixture was stirred and warmed to 50 C. for 4 hours. It was then cooled and the clear solution was added to 600 cc. of anhydrous ether. The resulting intermediate ester salt (diethyl riboflavyl phosphate hydrochloride) separated out as a gummy material and was washed with ether. The substance was then added to 50 cc. of water, whereupon solution occurred accompanied by a slight rise in temperature. Upon cooling, riboflavin phosphate crystallized there- 5 from, and was filtered, washed with 1 N I-ICl, ethanol and ether, and dried.

The following example will serve to illustrate the direct preparation of riboflavin phosphate without isolation of the intermediate ester salts or the lower alkyl riboflavyl phosphates:

EXAMPLE VIII To 73.2 cc. of phosphorus oxychloride (0.8 mole) were added with cooling to room temperature and stirring 64.8 cc. of methanol (1.6 moles). After evolution of I-ICl gas had subsided, the reaction product, which contained dimethyl monochlorophosphate, was allowed to stand for about 16 hours. 15.94 grams (0.04 mole) of riboflavin were then added. Solution took place in about 5 minutes. The resulting solution was stirred for 8 hours at room temperature. Then 50 cc. of water were slowly added, the temperature rising to 85 C. after which 100 cc. more of water were added. Crystallization of riboflavin monophosphoric acid ester occurred rapidly and was allowed to continue for about 16 hours. The product was then filtered, washed with 1 N HCl, ethanol and ether, and dried. The product thus obtained was essentially pure riboflavin phosphate.

We claim:

1. A process for manufacturing lower alkyl ri'boflavyl phosphates which comprises reacting riboflavin with a compound selected from the group consisting of a mono lower alkyl dichlorophosphate and a dilower alkyl monochloro-w phosphate.

2. A process for manufacturing monomethyl riboflavyl phosphate which comprises reacting riboflavin with monomethyl dichlorophosphate.

3. A process for manufacturing dimethyl riboflavyl phosphate which comprises reacting riboflavin with dimethyl monochlorophosphate.

4. A process for manufacturing monoethyl riboflavyl phosphate which comprises reacting riboflavin with monoethyl dichlorophosphate.

5. A process for manufacturing diethyl riboflavyl phosphate which comprises reacting riboflavin with diethyl monochlorophosphate.

6. A process which comprises reacting riboflavyl phosphate which comprises reacting riboa mono lower alkyl dichlorophosphate and a dilower alkyl monochlorophosphate and hydrolyzing the resulting reaction product so as to produce riboflavin phosphate.

7. A process which comprises reacting riboflavin with dimethyl monochlorophosphate and hydrolyzing the resulting reaction product so as to produce riboflavin phosphate.

8. A process which comprises reacting riboflavin with monomethyl dichlorophosphate and hydrolyzing the resulting reaction product so as to produce riboflavin phosphate.

9. A process which comprises hydrolyzing a compound selected from the group consisting of monoand dilower alkyl riboflavyl phosphates so as to produce riboflavin phosphate.

10. A process which comprises hydrolyzing monomethyl riboflavyl phosphate so as to produce riboflavin phosphate.

11. A process which comprises hydrolyzing dimethyl riboflavyl phosphate so as to produce riboflavin phosphate.

LEO A. FLEXSER. WAL'I'ER. G. FARKAS.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 1,945,183 Clemmensen Jan. 30, 1934 2,111,491 Kuhn et al Mar. 15, 1938 2,394,829 Whitehill et a1 Feb. 12, 1946 2,426,691 Jenkins Sept. 2, 1947 2,535,385 Breivogel Dec. 26, 1950 OTHER REFERENCES Kosolapofi, Organo Phosphorus Compounds, 1950, p. 223, pub. by John Wiley 8; Sons, Inc., New York. 

6. A PROCESS WHICH COMPRISES REACTING RIBOFLAVYL PHOSPHATE WHICH COMPRISES REACTING RIBOA MONO LOWER ALKYL DICHLOROPHOSPHATE AND A DILOWER ALKYL MONOCHLOROPHOSPHATE AND HYDROLYZING THE RESULTING REACTION PRODUCT SO AS TO PRODUCE RIBOFLAVIN PHOSPHATE. 